High morbidity and >20,000 deaths per year in the US alone are caused by infection with influenza virus. Neutralizing antibodies are one immune mechanism that provides effective protection against this virus. Natural virus-binding antibodies secreted prior to infection and virus-induced antibodies generated in the absence of T cell help, are both crucial for survival from this infection. The underlying basis of their induction however, is poorly understood. This proposal aims to define the basic principles that underlie the induction of these antibodies. Thus, providing a first step for identification of the precise molecular mechanisms that regulate their induction. The central hypothesis that will be tested is that intrinsic differences in B cell subsets contribute significantly to the regulation of antiviral humoral responses and that B-1 cells are one B cell subset participating early in immune defense. The first aim is to characterize distinct virus-specific B cell populations induced to influenza virus A/PR8 in mice and to determine their in vivo function. This will include the analysis of two distinct idiotype-expressing B cells, differing in the kinetics of their responses, and of the B-1 cell subset, which secretes virus-binding natural antibodies. These studies will test the hypothesis that virus-specific B cell subpopulations differ in lifespan, ability to proliferate and to differentiate to antibody-secreting cells. Virus-responding B cell subsets will be characterized by 10-color flow cytometry. Proliferative responses will be assessed by in vivo labeling of dividing cells. Antibody secretion will be measured by ELISA and ELISPOT of FACS-purified B cell subsets. Immunohistochemistry will determine the precise tissue location of idiotype-expressing B cells. The second aim is to test whether the function and fate of virus-specific B cells is determined by their intrinsic regulatory properties. For this, B cell subset responses will be characterized in T cell-deficient mice, as for aim #1. B cell receptor-affinity will be assessed to determine its influence on antibody-kinetics. Phenotypic studies are expected to identify new molecules involved in the regulation of early virus-specific B cell responses. The third aim is to test whether B cell differentiation into short-lived antibody-secreting cells causes the rapid disappearance of the early expressed idiotypes. Molecular outcomes of B cell subset activation will be assessed by real-time RT-PCR and Western-blot analysis of genes known to regulate B cell differentiation. This study challenges the current paradigm of B cell activation. Successful outcome of this study would provide a new framework that explains better how protective antiviral B cell responses are induced. It would also form the basis for defining the precise molecular mechanisms of early virus-specific B cell activation, thus for identifying possible new targets for rationale vaccine design.